Investigating myoblast-adipocyte crosstalk; influence on metabolic function and redox homeostasis

Background As life expectancy continues to increase globally, the incidence of age-related conditions is also rising. Among these, sarcopenic obesity (SO) has emerged as a growing concern and becoming an emerging health burden in ageing populations. SO is characterised by the coexistence of reduced skeletal muscle (SM) mass and increased adiposity, leading to impaired metabolic health and functional decline. Although SM and adipose tissue (AT) are increasingly recognised as endocrine organs that communicate through secreted factors, the precise mechanisms by which this crosstalk contributes to the pathogenesis of SO remain poorly understood. Identifying how secretomes from inflamed or metabolically challenged tissues affect the opposing cell type may provide new insight into the development of this condition and allow for an understanding of the pathogenesis of SO becoming known. Methods In this study, a bidirectional conditioned media (CM) model was created using murine C2C12 myoblasts and 3T3-L1 pre-adipocytes to assess how secreted factors under basal, insulin stimulated and LPS stimulated conditions influence metabolic function in the opposing cell line. CM was transferred between the cell types and multiple analysis was performed to determine metabolic function and redox homeostasis. Results Myoblasts exposed to CM from pre-adipocytes demonstrated marked alterations in mitochondrial function across all three treatment conditions. Proton leak was significantly increased in response to all CM conditions, while LPS CM specifically elevated mitochondrial membrane potential. All CM types increased hydroxyl radicals and mitochondrial superoxide. Structural analysis revealed a reduction in average mitochondrial branch length under each CM condition, consistent with increased fragmentation. At the transcriptional level, mitochondrial regulatory genes were upregulated in a treatment-dependent manner, with PGC1-α and COX4 elevated in response to insulin and LPS CM, while COX1 increased across all CM conditions. Inflammatory markers, including IL-6, were higher following exposure to insulin CM and LPS CM, indicating a shift towards a more pro-inflammatory state. Cytokine profiling showed reduced secretion of CCL2, IL-6 and CCL5 across all CM treatments, while GDF-15 levels were consistently increased. Conversely, pre-adipocytes treated with CM derived from myoblasts exhibited a parallel increase in inflammatory gene expression, including CCL2 and CCL5, across all three CM conditions. Genes regulating mitochondrial biogenesis, such as PGC-1α and TFAM, were consistently upregulated, whereas OPA1 expression was reduced in response to insulin, LPS and combined insulin + LPS CM. Functional assays demonstrated impaired mitochondrial respiration, with distinct patterns depending on the CM treatment. Non-mitochondrial respiration was altered predominantly by LPS CM, maximal respiration was reduced under both insulin and LPS CM, and proton leak was elevated in the LPS CM and insulin + LPS CM 9 groups. These functional changes were accompanied by increased mitochondrial content, particularly in response to insulin CM and insulin + LPS CM, and elevated MitoSOX signal in the insulin + LPS condition, indicative of greater superoxide production. Structural analysis further revealed reduced branch length and junction number most prominently following LPS CM and insulin + LPS CM, suggesting a more fragmented mitochondrial network. Cytokine analysis confirmed a pronounced inflammatory response, with CCL2, CXCL1, IL-6, CCL5 and GDF-15 markedly increased, especially under insulin + LPS CM. Conclusion Overall, the results show that secreted factors in CM alone are enough to drive metabolic dysfunction in both muscle and adipose cells. This dysfunction is marked by disrupted mitochondrial structure and function, as well as increased oxidative stress. The findings support the idea that unhealthy communication between these tissues could be a key contributor to the development of SO. Although processes such as inflammation, oxidative stress, and mitochondrial dysfunction are believed to play a role, the precise mechanisms connecting excess fat accumulation with muscle decline are not yet fully understood.